Spectroscopic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismutase and Fe-superoxide dismutase

We have compared the active sites of Escherichia coli Fe-substituted (Mn)superoxide dismutase [Fe-sub-(Mn)SOD] and Fe-SOD to elucidate the basis for the inactivity of Fe-sub-(Mn)SOD, despite its apparent similarity to Fe- SOD. The active site of (reduced) Fe²⁺-sub-(Mn)SOD is qualitatively similar to that of native Fe²⁺-SOD, indicating similar active site structures and coordination environments for Fe²⁺. Its nativelike pK is indicative of nativelike local electrostatics, and consistent with Fe²⁺-sub-(Mn)-SOD's retention of ability to reduce O₂^.- [Vance and Miller (1998) J. Am. Chem. Soc. 120(3), 461-467]. The active site of (oxidized) Fe³⁺-sub-(Mn)SOD differs from that of Fe³⁺-SOD with respect to the EPR signals produced at both neutral and high pH, indicating different coordination environments for Fe³⁺. Although Fe³⁺-sub-(Mn)SOD binds the small anions N₃^- and F^-, the K(D) for N₃^- is tighter than that of Fe³⁺-SOD, suggesting that the (Mn)SOD protein favors anion binding more than does the (Fe)SOD protein. The EPR spectral consequences of binding F^- are reminiscent of those observed upon binding the first F^- to Fe³⁺-SOD, but the EPR spectrum obtained upon binding N₃^- is different, consistent with crystallographic observation of a different binding mode for N₃^- in Themus thermophilus Mn-SOD than Fe-SOD [Lah, M., et al. (1995) Biochemistry 34, 1646-1660]. We find a pK of 8.5 to be associated with dramatic changes in the EPR spectrum. In addition, we confirm the pK between 6 and 7 that has previously been reported based on changes in the optical signal and N₃^- binding [Yamakura, F., et al. (1995) Eur. J. Biochem. 227, 700-706]. However, this latter pK appears to be associated with much subtler changes in the EPR spectrum. The non-native pKs observed in Fe³⁺-sub-(Mn)SOD and the differences in the Fe³⁺ coordination indicated by the EPR spectra are consistent with Fe³⁺-sub-(Mn)SOD's inability to oxidize O₂^.- and suggest that its low E°is due to perturbation of the oxidized state.